Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Intercellular communication analysis predicts IL-1β as a candidate upstream signal potentially associated with neutrophils. ( A ) Chord diagram showing predicted ligand–target interactions between sender cell populations and receiver SSPCs in the NonU microenvironment. Immune cell populations contributed prominently to the inferred SSPC-directed signaling network. ( B ) Dot plot showing the top predicted ligands received by SSPCs and their expression patterns across potential sender cell populations. Dot size indicates the percentage of ligand-expressing cells, and color indicates average expression. ( C ) Heatmap showing log-fold changes in prioritized ligands in their corresponding sender populations when comparing NonU with Frac conditions. ( D ) Heatmap showing the predicted regulatory potential of prioritized ligands on SSPC target genes. Candidate transcription factors are indicated. ( E ) GO enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis-related terms are highlighted. ( F ) KEGG enrichment analysis of predicted IL-1β target genes in SSPCs. Ferroptosis and fatty acid metabolism pathways are highlighted. Red arrows and red-colored labels indicate representative ligands, cell populations, target genes, transcription factors, or enriched terms highlighted and discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: IL-1β induces ferroptotic stress and impairs SSPC function in vitro. ( A ) Schematic overview of SSPC isolation by fluorescence-activated cell sorting and subsequent in vitro validation experiments. ( B ) Flow cytometry gating strategy for isolating Lin − /DPP4 + SSPCs from mouse callus tissue at 7 days post-fracture. ( C ) Representative Western blot images showing the expression of ACSL4, COX2, 4-HNE, NF-κB p65, and EPAS1 in SSPCs treated with vehicle, erastin, or IL-1β. β-actin was used as the loading control. ( D ) Quantification of Western blot protein expression levels shown in panel C ( n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Oxidized lipids are indicated by increased green fluorescence and reduced red fluorescence. Scale bar = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs. Scale bar = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining and the relative FerroOrange fluorescence intensity ( n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of colony-forming unit efficiency and cell viability measured by CCK-8 assay ( n = 3). ( I ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression ( n = 3). ( J ) Representative images of tri-lineage differentiation assays, including Alizarin Red S staining for osteogenesis, Alcian Blue staining for chondrogenesis, and Oil Red O staining for adipogenesis. Scale bar = 200 μm. ( K ) qRT-PCR analysis of osteogenic, chondrogenic, and adipogenic marker gene expression, including Runx2, Opn, Acan, Col2a1, Pparg, and Cebpa ( n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: In Vitro, Isolation, Fluorescence, FACS, Biomarker Discovery, Flow Cytometry, Western Blot, Expressing, Control, Staining, CCK-8 Assay, Quantitative RT-PCR, Marker, Gene Expression, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Pharmacological EPAS1 inhibition attenuates IL-1β-induced ferroptotic stress and differentiation impairment in primary SSPCs. ( A ) Representative images of Alcian Blue and Alizarin Red S staining in primary SSPCs treated with vehicle control, IL-1β, IL-1β plus PT2385, or PT2385 alone. Scale bars = 200 μm. ( B ) Representative Western blot images showing ACSL4, 4-HNE, EPAS1, and NF-κB p65 protein levels in SSPCs under the indicated treatments. GAPDH was used as the loading control. ( C ) qRT-PCR analysis of differentiation-related marker genes, including the chondrogenic markers Sox9 and Acan and the osteogenic markers Runx2 and Opn (n = 3). ( D ) Quantification of Western blot protein expression levels shown in panel ( B ) (n = 3). ( E ) Representative BODIPY 581/591 C11 fluorescence images showing lipid peroxidation in SSPCs. Red fluorescence indicates non-oxidized lipid signal, and green fluorescence indicates oxidized lipid signal. Scale bars = 50 μm. ( F ) Representative FerroOrange fluorescence images showing intracellular Fe 2+ levels in SSPCs under the indicated treatments. Scale bars = 50 μm. ( G ) Quantification of the red/green fluorescence intensity ratio from BODIPY 581/591 C11 staining (n = 3). A lower red/green ratio indicates increased lipid peroxidation. ( H ) Quantification of relative FerroOrange fluorescence intensity (n = 3). Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, Staining, Control, Western Blot, Quantitative RT-PCR, Marker, Expressing, Fluorescence, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: EPAS1 inhibition attenuates IL-1β-impaired bone regeneration in vivo. ( A ) Representative micro-CT three-dimensional reconstructions and micro-CT sectional images of fractured mouse femurs from vehicle control, IL-1β, IL-1β plus PT2385, and PT2385 alone groups at 28 days post-fracture. Scale bars = 1 mm. ( B ) Quantitative micro-CT analysis of bone volume (BV) and bone volume fraction (BV/TV) in the fracture callus region. ( C ) Quantitative histomorphometric analysis of bone area and callus area among the four treatment groups. ( D ) Representative Safranin O/Fast Green staining images of the fracture callus region showing callus organization, cartilage matrix, and newly formed bone tissue under the indicated treatments. Scale bars = 500 μm. Data are presented as mean ± SD; n = 6 mice per group. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Inhibition, In Vivo, Micro-CT, Control, Staining, Comparison

Journal: Current Issues in Molecular Biology

Article Title: IL-1β/EPAS1-Associated Ferroptotic Stress Impairs Skeletal Stem/Progenitor Cell Function in Inflammation-Associated Fracture Nonunion

doi: 10.3390/cimb48060606

Figure Lengend Snippet: Mendelian randomization provides exploratory genetic support linking IL-1β and EPAS1 to human bone nonunion risk. ( A ) Forest plot summarizing inverse-variance weighted Mendelian randomization estimates for the tested exposures and bone nonunion risk. Odds ratios and 95% confidence intervals are shown. ( B ) Scatter plots showing the associations between SNP effects on EPAS1 expression or IL-1β levels and SNP effects on bone nonunion risk. Lines indicate estimates from different Mendelian randomization methods. ( C ) Single-SNP forest plots showing individual SNP estimates for EPAS1 and IL-1β. The combined inverse-variance weighted estimates are shown at the bottom. ( D ) Leave-one-out analyses for EPAS1 and IL-1β, performed by sequentially excluding one SNP at a time. ( E ) Funnel plots used to assess potential directional pleiotropy in the Mendelian randomization analyses for EPAS1 and IL-1β. Red colors, red lines, and numbers indicate representative annotations used to highlight key features discussed in the main text.

Article Snippet: Cells were treated with recombinant mouse IL-1β (10 ng/mL), Ferrostatin-1 (Fer-1; MedChemExpress, Monmouth Junction, NJ, USA; #HY-100579; 1 μM), or their combination for 24 h. Cells were then collected for Western blot analysis of ferroptosis-related proteins and, where indicated, FerroOrange staining to assess intracellular ferrous iron accumulation.

Techniques: Expressing

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA inhibits the secretion of inflammatory factors and glycolysis in macrophages (A and B) Schematic diagram of THP-1 cell (A) and BMDMs (B) activation into pro-inflammatory macrophages. Created with BioRender.com. (C) The concentrations of IL-6, TNF-α, and IL-1β in THP-1 cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (D) The concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with different concentrations of 3-HPA (0, 0.625, 1.25, 5 mM) followed by LPS stimulation. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (E) The concentrations of IL-6, TNF-α, and IL-1β in BMDM cell supernatants were quantified by ELISA after 24 h treatment with LPS (100 ng/mL) or LPS+3-HPA (5 mM). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (F) Bubble plot of KEGG pathway enrichment analysis for differentially expressed genes between LPS (100 ng/mL) and LPS+3-HPA (5 mM) treated in THP-1 cells. The size of each bubble represents the number of differentially expressed genes, and the color indicates the enrichment factor. (G and H) Pyruvate and lactate levels in THP-1 cells (G) and BMDMs (H) treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM). (I) Immunoblots of protein expression levels of HK, GAPDH, PKM, and LDHA in THP-1 cells treated with LPS (100 ng/mL), or LPS+3-HPA (5 mM), and quantitative results of GAPDH. (J) The mRNA levels of GAPDH in THP-1 cells treated with LPS (100 ng/mL) or LPS+3-HPA (5 mM). (K) GAPDH activity assay in THP-1 cells and BMDMs treated with PBS, LPS (100 ng/mL), or LPS+3-HPA (5 mM). Data in (G–K) are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Mouse IL-1β Precoated ELISA Kit , Dakewe , 1210122.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Activity Assay

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: 3-HPA enhances the metabolite content of the TCA cycle and mitochondrial oxidation (A) Schematic diagram of THP-1 with 2-DG treatment. Created with BioRender.com. (B) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS, LPS+3-HPA, LPS+2-DG, or LPS+3-HPA+2-DG. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001; ns, not significant. (C) Schematic diagram of THP-1 with high glucose treatment. Created with BioRender.com. (D) Concentrations of IL-6, TNF-α, and IL-1β in supernatants of THP-1 cells treated with LPS+3-HPA, LPS+3-HPA+glucose. Data are the means ± SD and n = 3 per group. Statistical significance was determined using unpaired Student’s t test with ∗∗∗p < 0.001; ns, not significant. (E) KEGG pathway enrichment analysis of metabolic pathways in BMDM cells treated with LPS or LPS+3-HPA. (F) Relative abundance of metabolite (ornithine, citrulline, L-malate, succinic acid, trans-aconitic acid, cis-aconitic acid) in BMDM cells treated with LPS or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using unpaired Student’s t test with ∗p < 0.05; ∗∗p < 0.01. (G) Correlation network of metabolites and genes in the metabolic pathway. Nodes represent metabolites (blue squares) and genes (colored circles). Gray edges indicate pairwise correlations between metabolites and genes. The colors similarly represent expression levels, with red typically indicating higher expression and blue indicating lower expression compared to the mean. (H) Schematic diagram of arginine metabolism and the TCA cycle. (I) Heatmap of mitochondrial oxidation-related gene expression associated with differentially expressed metabolites. Red indicates relatively high gene expression, while blue indicates relatively low gene expression within each row. (J) Schematic diagram of the catalytic function of the GAPDH enzyme. (K) Concentrations of the NAD + /NADH ratio in THP-1 cells and BMDM cells treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001; (L) ATP concentrations in THP-1 cells and BMDMs treated with PBS, LPS, or LPS+3-HPA. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗ p < 0.05; ∗∗∗∗ p < 0.0001; ns, not significant. (M) Representative images and mitochondrial analysis of BMDM cells treated with PBS, LPS, or LPS+3-HPA. Scale bars, 1 μm (upper) and 0.25 μm (lower). For mitochondrial number, n = 8–12 ( n = 12 for PBS, n = 8 for LPS, n = 9 for LPS+3-HPA group).

Article Snippet: Mouse IL-1β Precoated ELISA Kit , Dakewe , 1210122.

Techniques: Expressing, Gene Expression

Journal: iScience

Article Title: 3-Hydroxypropionic acid converts inflammatory macrophage glycolysis into mitochondrial oxidation through GAPDH carboxyethylation

doi: 10.1016/j.isci.2026.116258

Figure Lengend Snippet: GAPDH carboxyethylation inhibited macrophage glycolysis and the release of inflammatory factors (A) Schematic workflow illustrating the strategy of silencing endogenous GAPDH via 3′UTR-targeting siRNA and overexpressing exogenous GAPDH. (B) Immunoblot and quantitative analysis of GAPDH protein in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗p < 0.01. (C) Relative mRNA expression of GAPDH in 293 T cells transfected with GAPDH 3′UTR-targeting siRNAs (siGAPDH 1, siGAPDH 2) or siRNA NC. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test ∗∗∗∗p < 0.0001. (D) Immunoblot analysis of FLAG-tagged exogenous GAPDH(E) and GAPDH in 293 T cells. Knockdown of endogenous GAPDH with siRNA followed by the overexpression of GAPDH (E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test with ∗p < 0.05; ns, not significant. (E) Relative mRNA expression of GAPDH in 293 T cells knockdowned endogenous GAPDH (siGAPDH) and overexpressed GAPDH (C) and GAPDH (E), respectively. Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (F) GAPDH activity assay in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗∗∗p < 0.0001; ns, not significant. (G) Concentrations of lactate and pyruvate in 293 T cells transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C), GAPDH(E). Data are the means ± SD and n = 4 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗∗p < 0.0001. (H) Relative mRNA expression of IL-6 , TNF-α , and IL-1β in THP-1 cells which transfected with GAPDH 3′UTR siRNA, and overexpressing GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (I) The concentration of TNF-α in THP-1 cells that overexpressed GAPDH(C) or GAPDH(E). Data are the means ± SD and n = 3 per group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test with ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant.

Article Snippet: Mouse IL-1β Precoated ELISA Kit , Dakewe , 1210122.

Techniques: Western Blot, Transfection, Expressing, Knockdown, Over Expression, Activity Assay, Concentration Assay